Methods and compositions for inhibiting angiogenesis

ABSTRACT

The present invention relates to methods and compositions for modulating angiogenesis. In particular, the present invention relates to Pigment Epithelial-derived Factor (PEDF) fragments for use in modulating angiogenesis and treating angiogenesis mediated disease.

The present application claims priority to U.S. Provisional ApplicationSer. No. 60/986,124, filed Nov. 7, 2007, which is herein incorporated byreference in its entirety.

This invention was made with government support under grant numberW81XWH-0-6-1-0103 awarded by the Army/MRMC and grant number R01HL068033awarded by the National Institutes of Health. The government has certainrights in the invention.

FIELD OF THE INVENTION

The present invention relates to methods and compositions for modulatingangiogenesis. In particular, the present invention relates to PigmentEpithelial-derived Factor (PEDF) fragments for use in modulatingangiogenesis and treating angiogenesis mediated disease.

BACKGROUND OF THE INVENTION

Tumor angiogenesis is a complex process in which new blood vessels areformed in response to interactions between tumor cells and endothelialcells (ECs), growth factors, and extracellular matrix components. Tumorvessels promote growth and progression of human solid tumors (e.g.,cancer of the liver, bladder, and prostate). New tumor blood vesselspenetrate into cancerous growths, supplying nutrients and oxygen andremoving waste products (Jung et al., 2002; Folk-man, 2002; Kerbel andKamen, 2004; Stupack and Cheresh, 2004). A large number of studies havedemonstrated that tumor cells secrete angiogenic growth factors tostimulate EC proliferation and to induce angiogenesis. Among them,vascular endothelial growth factor (VEGF) is one of the most potentangiogenic factors, and it is overexpressed in many human cancers (Junget al., 2002).

Targeting VEGF for human cancer therapy has shown some promise in thetreatment of colorectal cancer, demonstrating the potential for cancertherapy based upon blocking angiogenesis (Ferrara et al., 2004).However, targeting VEGF for human cancer therapy has not been successfulin a multiplicity of other tumor types, suggesting that other factors orcomponents also play a critical role in tumor angiogenesis (Jung et al.,2002; Kerbel and Kamen, 2004). The identification of these factors andcomponents have important implications in human cancer therapy.

Thus, there is need for the identification of other factors orcomponents that are involved in tumor angiogenesis. Furthermore, newcompositions and methods are required to target these factors that canbe used to treat cancer (e.g., to inhibit angiogenesis, whose loss isassociated with cancer).

SUMMARY OF THE INVENTION

The present invention relates to methods and compositions for modulatingangiogenesis. In particular, the present invention relates to PigmentEpithelial-derived Factor (PEDF) fragments for use in modulatingangiogenesis and treating angiogenesis mediated disease.

In some embodiments, the present invention provides compositionscomprising an isolated peptide, wherein the peptide consists of an aminoacid sequence selected from: SEQ ID NOs:2, 12, 13, 22, 23, 33, 34, 41,42, 43-49, 52, 53, 62, 63, 73, 74, 84, 85, and 92-100. In otherembodiments, the peptide comprises, or consists of, SEQ ID NOs: 1-49 and52-100. In certain embodiments, one or more of the amino acid in any ofthese sequences are substituted with modified versions of theappropriate amino acid or amino acids that do not detrimentally affectthe anti-angiogenic activity of the peptide.

In some embodiments, the present invention provides methods ofinhibiting angiogenesis, comprising: contacting a tissue (e.g.,exhibiting angiogenesis) with a composition comprising an isolatedpeptide (e.g., under conditions such that angiogenesis is decreased inthe tissue), wherein the peptide comprises, or consists of, an aminoacid sequence selected from: SEQ ID NOs:2, 12, 13, 22, 23, 33, 34, 41,42, 43-49, 52, 53, 62, 63, 73, 74, 84, 85, and 92-100. In certainembodiments, the peptide comprises, or consists of, SEQ ID NOs: 1-49 and52-100. In particular embodiments, the methods further comprise the stepof administering a second agent to the tissue (e.g., anti-cancer agentor anti-angiogenic agent). In particular embodiments, the tissue iscancerous tissue. In further embodiments, the tissue is in a subject. Inparticular embodiments, the composition is administered to the subjectat a dosage of between 5-150 mg/kg or between 10-75 mg/kg (e.g., 10mg/kg . . . 25 mg/kg . . . 50 mg/kg . . . 60 mg/kg . . . 75 mg/kg . . .100 mg/kg . . . 125 mg/kg . . . or 150 mg/kg). In certain embodiments,the subject has ocular neovascularization or cancer.

In certain embodiments, the peptide exhibits anti-angiogenic oranti-cancer activity. In particular embodiments, the peptide consists of(or consists essentially of) the amino acid sequence shown in SEQ IDNO:2 or SEQ ID NO:52.

In further embodiments, the compositions further comprise a non-aminoacid chemical moiety, wherein the non-amino acid chemical moiety isattached to, or associated with, the peptide. In certain embodiments,the chemical moiety is a fluorescent compound or a compound intended toaid in the in vivo delivery of the peptide.

In some embodiments, the compositions further comprise an anti-canceragent different from the peptide. In certain embodiments, theanti-cancer agent is selected from the group consisting of: rapamycin,Alkylating agents (e.g., Cisplatin and carboplatin, oxaliplatin,mechlorethamine, cyclophosphamide, chlorambucil), Anti-metabolites(e.g., azathioprine, or mercaptopurine), Plant alkaloids and terpenoids(e.g., vinca alkaloids and taxanes), Vinca alkaloids (e.g, Vincristine,Vinblastine, Vinorelbine, and Vindesine), Podophyllotoxin, Taxanes(e.g., paclitaxel), Topoisomerase inhibitors (e.g., camptothecins:irinotecan and topotecan, amsacrine, etoposide, etoposide phosphate, andteniposide), Antitumour antibiotics (e.g., dactinomycin), and Monoclonalantibodies (e.g., trastuzumab, cetuximab, rituximab, and Bevacizumab).

In particular embodiments, the compositions further comprise aphysiological tolerable buffer. In other embodiments, the compositionsfurther comprise an anti-angiogenic agent different from the peptide. Incertain embodiments, the anti-angiogenic agent is selected from thegroup consisting of: soluble VEGFR-1, NRP-1, Angiopoietin 2, TSP-1,TSP-2, angiostatin and related molecules, endostatin, vasostatin,calreticulin, platelet factor-4, TIMP, CDAI, Meth-1, Meth-2, IFN-α, -βand -γ, CXCL10, IL-4, IL-12 IL-18, prothrombin (kringle domain-2),antithrombin III fragment, prolactin, VEGI, SPARC, osteopontin, maspin,canstatin, proliferin-related protein, and restin. In particularembodiments, the compositions are pharmaceutical compositions (e.g.,suitable for injection into an animal or a human).

In some embodiments, the present invention provides a compositioncomprising an isolated fragment of pigment epithelial-derived factor(PEDF) (e.g., a fragment comprising or consisting of: SEQ ID NO:2, SEQID NO:1, SEQ ID NOs:3-49, SEQ ID NOs:52-100, or mimetics, variants, orderivatives thereof), wherein the fragment of PEDF exhibitsanti-angiogenic activity. In other embodiments, the present inventionprovides a composition comprising an isolated peptide comprising, orconsisting of, or consisting essentially of: SEQ ID NOs:2, 12, 13, 22,23, 33, 34, 41-49, 52, 53, 62, 63, 73, 74, 84, 85, and 92-100, orcombinations thereof (e.g., wherein the peptide has anti-angiogenicactivity). In some embodiments, the composition further comprises asecond fragment of PEDF (e.g., a fragment comprising or consisting ofamino acids 78-121 of PEDF). In some embodiments, the compositionfurther comprises additional agents with anti-angiogenesis oranti-cancer activity. In some embodiments, the composition is apharmaceutical composition.

The present invention further provides a method of inhibitingangiogenesis, comprising: contacting a tissue exhibiting angiogenesiswith a fragment of PEDF under conditions such that angiogenesis isdecreased in the tissue. In some embodiments, the fragment comprises orconsists of: SEQ ID NO:2, SEQ ID NO:1, SEQ ID NOs:3-49, SEQ IDNOs:52-100, or mimetics, variants, or derivatives thereof. In furtherembodiments, the peptide comprises, or consists of, or consistsessentially of: SEQ ID NOs:2, 12, 13, 22, 23, 33, 34, 41-49, 52, 53, 62,63, 73, 74, 84, 85, and 92-100 or combinations thereof (e.g., whereinthe peptide has anti-angiogenic activity). In some embodiments, themethod further comprises the step of administering a second agent to thetissue. In some embodiments, the second agent is a second fragment ofPEDF (e.g., comprising or consisting of amino acids 78-121 of PEDF) or aknown anti-angiogenic or anti-cancer agent. In some embodiments, thetissue is cancerous tissue. In some embodiments, the tissue is in asubject.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows a schematic representation of identified functional domainsof PEDF, including major phosphorylation sites and domains known to becritical for interactions with extracellular matrix.

FIG. 2 shows peptide design for the mapping of the 34-meranti-angiogenic fragments.

FIG. 3 shows the results of an endothelial cell chemotaxis assay, whereendothelial cells traversed a gelatinized microporous membrane up agradient of bFGF in the absence and the presence of PEDF peptides,including a 14-mer (SEQ ID NO:1; FIG. 3A), 18-mer (SEQ ID NO:2; FIG.3B), and 23-mer (SEQ ID NO:3; FIG. 3C).

FIG. 4 shows that PEDF peptides block survival effect of bFGF byinducing endothelial cells apoptosis. FIG. 4A shows immunofluorescentstaining of human endothelial cells. Confluent HMVEC cells growing oncoverslips were incubated overnight in low serum media (0.2% FBS inMCDB)±PEDF peptides in the presence or absence of bFGF. ApopTagFluorescein kit (Chemicon) was used to determine apoptotic cells. FIG.4B shows quantitative analysis of apoptosis. 3-5 fields (10×) wereanalyzed with epi-fluorescent microscope, and cells were counted usingMetaMorph software. Apoptotic (FITC positive) cells were calculated as a% of total cells stained with PI per field.

FIG. 5 shows the in vivo anti-angiogenic effect of the PEDF peptides.FIGS. 5A and 5B show the results of a corneal angiogenesis assay. The 34mer and 18-mer peptides were incorporated with bFGF (50 ng/pellet) intoslow-release sucralfate pellets, which were surgically mplanted into thecornea of anesthetized mice, 0.5-1 mm from the vascular limbus. Theresponses were scored on day 5 post implantation and the ingrowth ofblood vessels from the cornea to the pellet was considered a positiveresponse. The responses were scored as positive corneas of totalimplanted: statistical significance was evaluated with Fisher's Exacttest. P<0.05 was considered significant. FIG. 5A shows photographs ofrepresentative corneas. FIG. 5B shows tabulated results of the corneaassay. FIGS. 5C and 5D shows the directed in vivo angiogenesis assay(DIVAA) for the 34-mer, 23-mer, and 18-mer peptides. The peptides wereincorporated with a mix of bFGF and VEGF (37.5 and 12.5 ng/ml,respectively) into angioreactors filled with matrigel. The reactors wereimplanted s.c onto the flanks of the nude mice. On day 7, The reactorswere harvested and photographed (5C). Endothelial cells collected fromimplants by dilution/centrifugation, stained with FITC-lectin andquantified by flow cytometry (5D). In both assays the 18-mer showed thebest anti-angiogenic characteristics. The 23-mer failed to inhibitangiogenesis in the DIVAA assay.

FIG. 6 shows the effects of 34-mer and 18-mer systemic treatment ontumor growth.

FIG. 7 shows the amino acid sequence of a full-length PEDF peptide (SEQID NO:50), which is accession number AAP36928.

FIG. 8 shows the growth of orthotopic renal cancer xenografts in thepresent of control peptide (scrambled peptide, SEQ ID NO:51), P18(18-mer peptide, SEQ ID NO:2), rapamycin (R) and the combination ofrapamycin and P18.

FIG. 9 shows metastases formation by orthotopic renal cancer xenograftsas described in Example 1. In this figure: Control=scrambled peptide,P18=18mer (SEQ ID NO:2), R=rapamycin. The incidence (affected organs) isshown in the left upper corner.

FIG. 10 shows the amino acid sequence of a full-length PEDF peptide (SEQID NO:101), which is accession number AAH13984.

DEFINITIONS

As used herein, the term “subject diagnosed with a cancer” refers to asubject who has been tested and found to have cancerous cells. Thecancer may be diagnosed using any suitable method, including but notlimited to, biopsy, x-ray, blood test, and the diagnostic methods of thepresent invention. A “preliminary diagnosis” is one based only on visual(e.g., CT scan or the presence of a lump) and antigen tests.

As used herein, the term “effective amount” refers to the amount of acomposition (e.g., a PEDF peptide) sufficient to effect beneficial ordesired results. An effective amount can be administered in one or moreadministrations, applications or dosages and is not intended to belimited to a particular formulation or administration route.

As used herein, the term “administration” refers to the act of giving adrug, prodrug, or other agent, or therapeutic treatment (e.g.,compositions of the present invention) to a subject (e.g., a subject orin vivo, in vitro, or ex vivo cells, tissues, and organs). Exemplaryroutes of administration to the human body can be through the eyes(ophthalmic), mouth (oral), skin (transdermal), nose (nasal), lungs(inhalant), oral mucosa (buccal), ear, by injection (e.g.,intravenously, subcutaneously, intratumorally, intraperitoneally, etc.)and the like.

As used herein, the term “co-administration” refers to theadministration of at least two agent(s) (e.g., a PEDF peptide and one ormore other agents such as an anti-cancer agent) or therapies to asubject. In some embodiments, the co-administration of two or moreagents or therapies is concurrent. In other embodiments, a firstagent/therapy is administered prior to a second agent/therapy. Those ofskill in the art understand that the formulations and/or routes ofadministration of the various agents or therapies used may vary. Theappropriate dosage for co-administration can be readily determined byone skilled in the art. In some embodiments, when agents or therapiesare co-administered, the respective agents or therapies are administeredat lower dosages than appropriate for their administration alone. Thus,co-administration is especially desirable in embodiments where theco-administration of the agents or therapies lowers the requisite dosageof a potentially harmful (e.g., toxic) agent(s).

As used herein, the term “toxic” refers to any detrimental or harmfuleffects on a subject, a cell, or a tissue as compared to the same cellor tissue prior to the administration of the toxicant.

As used herein, the term “pharmaceutical composition” refers to thecombination of an active agent (e.g., a PEDF peptide) with a carrier,inert or active, making the composition especially suitable fordiagnostic or therapeutic use in vitro, in vivo or ex vivo.

The terms “pharmaceutically acceptable” or “pharmacologicallyacceptable,” as used herein, refer to compositions that do notsubstantially produce adverse reactions, e.g., toxic, allergic, orimmunological reactions, when administered to a subject.

As used herein, the term “topically” refers to application of thecompositions of the present invention to the surface of the skin andmucosal cells and tissues (e.g., alveolar, buccal, lingual, masticatory,or nasal mucosa, and other tissues and cells that line hollow organs orbody cavities).

As used herein, the term “pharmaceutically acceptable carrier” refers toany of the standard pharmaceutical carriers including, but not limitedto, phosphate buffered saline solution, water, emulsions (e.g., such asan oil/water or water/oil emulsions), and various types of wettingagents, any and all solvents, dispersion media, coatings, sodium laurylsulfate, isotonic and absorption delaying agents, disintrigrants (e.g.,potato starch or sodium starch glycolate), and the like. Thecompositions also can include stabilizers and preservatives. Forexamples of carriers, stabilizers and adjuvants. (See e.g., Martin,Remington's Pharmaceutical Sciences, 15th Ed., Mack Publ. Co., Easton,Pa. (1975), incorporated herein by reference).

As used herein, the term “pharmaceutically acceptable salt” refers toany salt (e.g., obtained by reaction with an acid or a base) of acompound of the present invention that is physiologically tolerated inthe target subject (e.g., a mammalian subject, and/or in vivo or exvivo, cells, tissues, or organs). “Salts” of the compounds of thepresent invention may be derived from inorganic or organic acids andbases. Examples of acids include, but are not limited to, hydrochloric,hydrobromic, sulfuric, nitric, perchloric, fumaric, maleic, phosphoric,glycolic, lactic, salicylic, succinic, toluene-p-sulfonic, tartaric,acetic, citric, methanesulfonic, ethanesulfonic, formic, benzoic,malonic, sulfonic, naphthalene-2-sulfonic, benzenesulfonic acid, and thelike. Other acids, such as oxalic, while not in themselvespharmaceutically acceptable, may be employed in the preparation of saltsuseful as intermediates in obtaining the compounds of the invention andtheir pharmaceutically acceptable acid addition salts.

Examples of bases include, but are not limited to, alkali metal (e.g.,sodium) hydroxides, alkaline earth metal (e.g., magnesium) hydroxides,ammonia, and compounds of formula NW₄ ⁺, wherein W is C₁₋₄ alkyl, andthe like.

Examples of salts include, but are not limited to: acetate, adipate,alginate, aspartate, benzoate, benzenesulfonate, bisulfate, butyrate,citrate, camphorate, camphorsulfonate, cyclopentanepropionate,digluconate, dodecylsulfate, ethanesulfonate, fumarate, flucoheptanoate,glycerophosphate, hemisulfate, heptanoate, hexanoate, chloride, bromide,iodide, 2-hydroxyethanesulfonate, lactate, maleate, methanesulfonate,2-naphthalenesulfonate, nicotinate, oxalate, palmoate, pectinate,persulfate, phenylpropionate, picrate, pivalate, propionate, succinate,tartrate, thiocyanate, tosylate, undecanoate, and the like. Otherexamples of salts include anions of the compounds of the presentinvention compounded with a suitable cation such as Na⁺, NH₄ ⁺, and NW₄⁺ (wherein W is a C₁₋₄ alkyl group), and the like. For therapeutic use,salts of the compounds of the present invention are contemplated asbeing pharmaceutically acceptable. However, salts of acids and basesthat are non-pharmaceutically acceptable may also find use, for example,in the preparation or purification of a pharmaceutically acceptablecompound.

For therapeutic use, salts of the compounds of the present invention arecontemplated as being pharmaceutically acceptable. However, salts ofacids and bases that are non-pharmaceutically acceptable may also finduse, for example, in the preparation or purification of apharmaceutically acceptable compound.

As used herein, the term “sample” is used in its broadest sense. In onesense, it is meant to include a specimen or culture obtained from anysource, as well as biological and environmental samples. Biologicalsamples may be obtained from animals (including humans) and encompassfluids, solids, tissues, and gases. Biological samples include bloodproducts, such as plasma, serum and the like. Environmental samplesinclude environmental material such as surface matter, soil, water,crystals and industrial samples. Such examples are not however to beconstrued as limiting the sample types applicable to the presentinvention.

DESCRIPTION OF THE INVENTION

Angiogenesis is the fundamental process by which new blood vessels areformed. The process involves the migration of vascular endothelial cellsinto tissue followed by the condensation of such endothelial cells intovess. Angiogenesis may be induced by an exogenous angiogenic agent ormay be the result of a natural condition. The process is essential to avariety of normal body activities such as reproduction, development andwound repair. Although the process is not completely understood, itinvolves a complex interplay of molecules that stimulate and moleculesthat inhibit the growth and migration of endothelial cells, the primarycells of the capillary blood vessels. Under normal conditions, thesemolecules appear to maintain the microvasculature in a quiescent state(i.e., without capillary growth) for prolonged periods which can lastfor several years or even decades. The turnover time for an endothelialcell is about one thousand days. However, under appropriate conditions(e.g., during wound repair), these same cells can undergo rapidproliferation and turnover within a much shorter period, and a turnoverrate of five days is typical under these circumstances. (See, e.g.,Folkman and Shing, 1989, J. Biol. Chem. 267(16):10931-10934; Folkman andKlagsbrun, 1987, Science 235:442-447).

Although angiogenesis is a highly regulated process under normalconditions, many diseases (characterized as “angiogenic diseases”) aredriven by persistent unregulated angiogenesis. In such disease states,unregulated angiogenesis can either cause a particular disease directlyor exacerbate an existing pathological condition. For example, ocularneovascularization has been implicated as the most common cause ofblindness and underlies the pathology of approximately twenty diseasesof the eye. In certain previously existing conditions such as arthritis,newly formed capillary blood vessels invade the joints and destroycartilage. In diabetes, new capillaries formed in the retina invade thevitreous humor and bleed, causing blindness. The compositions andmethods of the present invention may employed to treat such angiogenicdiseases.

Both the growth and metastasis of solid tumors are alsoangiogenesis-dependent (See, e.g., Folkman, 1986, J. Cancer Res.46:467-473; Folkman, 1989, J. Nat. Cancer Inst. 82:4-6; Folkman et al.1995, “Tumor Angiogenesis,” Chapter 10, pp. 206-32, in The MolecularBasis of Cancer, Mendelsohn et al., eds. (W. B. Saunders)). It has beenshown, for example, that tumors which enlarge to greater than about 2 mmin diameter must obtain their own blood supply and do so by inducing thegrowth of new capillary blood vessels. After these new blood vesselsbecome embedded in the tumor, they provide nutrients and growth factorsessential for tumor growth as well as a means for tumor cells to enterthe circulation and metastasize to distant sites, such as liver, lung orbone (See, e.g., Weidner 1991, New Eng. J. Med. 324(1):1-8). When usedas drugs in tumor-bearing animals, natural inhibitors of angiogenesiscan prevent the growth of small tumors (See, e.g., O'Reilly et al.,1994, Cell 79:315-328). Indeed, in some protocols, the application ofsuch inhibitors leads to tumor regression and dormancy even aftercessation of treatment (See, e.g., O'Reilly et al., 1997, Cell88:277-285). Moreover, supplying inhibitors of angiogenesis to certaintumors can potentiate their response to other therapeutic regimens(e.g., chemotherapy) (See, e.g., Teischer et al., 1994, Int. J. Cancer57:920-925). The compositions and methods of the present invention maybe used to treat cancer (e.g., by inhibiting tumorigenesis).

Although several angiogenesis inhibitors are currently under developmentfor use in treating angiogenic diseases (See, e.g., Gasparini, 1996,Eur. J. Cancer 32A(14):2379-2385), there are disadvantages associatedwith these proposed inhibitory compounds. For example, suramin is apotent angiogenesis inhibitor, but, at doses required to reach antitumoractivity, causes severe systemic toxicity in humans. Other compounds,such as retinoids, interferons and antiestrogens appear safe for humanuse but have only a weak anti-angiogenic effect. Still other compoundsmay be difficult or costly to make. In addition, the simultaneousadministration of several different inhibitors of angiogenesis may beneeded for truly effective treatment.

I. PEDF Mapping

PEDF is a major angiogenesis inhibitor in the eye. It is secreted at bythe retinal pigment epithelium (RPE) and regulated by oxygen levels.PEDF is responsible for the neurotrophic activity secreted by RPE cells.It differentiates retinoblastoma tumor cells and promotes survival ofthe more differentiated neuronal cells such as cerebellar granuleneurons, hippocampal neurons, photoreceptors, and cultured retinalneurons.

The complications of diabetes mellitus, kidney failure and loss ofvision involve major vascular abnormalities. Vascular beds in theposterior eye, namely the choroid and the retina, are quiescent innormal adult eye. DR starts with the damage to the small vessels in theretina. Decreased flow causes hypoxia and increases vascular endothelialgrowth factor (VEGF), causing inappropriate neovascularization andvascular leakage, whereas aberrant capillaries to invade the retina andthe vitreous humor (proliferative retinopathy) leading to hemorrhage,scarring, and detachment. The compositions and methods of the presentinvention may be used to treat such conditions.

VEGF is one of the most prevalent angiogenic growth factors, responsiblefor pathological angiogenesis in cancer and in eye disease, includingacute macular degeneration (AMD, wet type), diabetic retinopathy (DR)and retinopathy of prematurity (ROP). PEDF depletion makes cornea,vireous, retina, and choroid permissive for angiogenesis. In contrast toVEGF, PEDF remains high in normoxia and decreases in low O₂. Animalmodels of DR, ROP and AMD reveal that neovascularization in adult eye isdetermined by the ratio between pro-angiogenic VEGF and anti-angiogenicPEDF. Purified, adenoviral and lentiviral PEDF are effective againstneovascularization in animal models of DR and AMD.

The PEDF gene is mapped to 17p13, a locus frequently lost in primitivetumors of the CNS and in some ovarian tumors where PEDF is a candidatetumor suppressor. Genome analysis of mouse B16 melanoma and normal skinreveals frequent allelic loss of PEDF indicating a link between PEDF,melanoma and angiogenesis. PEDF treatment inhibits the growth ofendometrial carcinoma cells. PEDF secretion is low in senescentendometrial fibroblasts, a decrease that may contribute to theage-related increase in cancer incidence by creating permissiveenvironment for the endometrial tumors.

Crawford and co-workers showed that PEDF from Schwann cellsdifferentiates adjacent neuroblastoma tumors and acts as amultifunctional anti-tumor agent, by (a) inhibiting angiogenesis and (b)expanding Schwannian and differentiated components the tumor, creating afeedback loop that limits or reverses tumor growth. PEDF gene transferof inhibits tumor growth of thoracic malignancies, melanoma andhepatocellular carcinoma in syngeneic murine models.

PEDF belongs to the sub-family of inactive serpins (serine proteaseinhibitors), serpin reactive loop contributes neither to angioinhibitorynor to neurotropic function. PEDF acts against wide variety ofangiogenic stimuli by inducing apoptosis in the activated endothelialcells via a cascade where CD95L, a death ligand is upregulated by PEDFitself and CD95/Fas death receptor is increased by angiogenic stimuli.In addition, PEDF blocks cFLIP, an endogenous caspase inhibitor.

In contrast, PEDF induces survival of the neural crest cells andphotoreceptors. The region responsible for this neurotrophic functionhas been mapped to a 44-mer peptide (positions 58-101). PEDFneuroprotective function requires the activation of NF-κB transcriptionfactor and reverses the decrease in Bcl-2 in pericytes exposed toadvanced glycation end product (AGE).

PEDF is a 50 kDa glycoprotein. Numerous investigations have shed lighton the structure-function relationships of this molecule (FIG. 1). Itbinds to collagen and heparin, through nonoverlapping regions (collagenbinding may be required for PEDF antiangiogenic activity). PEDFC-terminal fragment (amino acids 195-400) does not reproduce the effectson self-renewal, angiogenesis or neuroprotection. The antiangiogenic andneurotrophic activities are located in the N-terminal portion of themolecule, in two sites. One anti-angiogenic region is clearly distinctthe neurotrophic epitope, it is a 34-amino acid (residues 44-77). Theother anti-angiogenic region is a small subdomain of the 44-mer, whichacid region that can mediate neurotrophic actions (amino acids 78-121).As a 44-mer this region is not anti-angiogenic, however a smallerfragment (ERT, residues 78-121) retains both neurotrophic and angiogenicactivity. The present invention is not limited to a particularmechanism. Nonetheless, taken together, the existing data support amodel in which binding of PEDF to extracellular matrix (collagen)regulates the exposure of molecular domains, and thus determine itsneurotrophic or antiangiogenic activities. In support of the notion thatallosteric modulation of PEDF can regulate its biological activity,phosphorylation of residues 24 and 114 by casein kinase 2 (CK2) canenhance the antiangiogenic functions and decrease neurotrophic activity.Conversely, phosphorylation at residue 227 by PKA decreases theantiangiogenic activity.

The combination of neuroprotective and antiangiogenic properties makesPEDF and active fragments thereof valuable agents in diseases anddisorders where neurovascular interactions go haywire, such as braintumors or macular degeneration; in the latter, choroidal blood vesselsinvade the delicate retinal macula, destroying high-acuity centralvision in their wake and leaving approximately half a million people inthe US legally blind. The compositions and methods of the presentinvention may be employed to treat such conditions.

PEDF further finds use in the treatment of highly vascular cancer types(e.g., breast cancer, ovarian cancer), highly metastatic cancer types(e.g., melanoma) and ocular disease (e.g., diabetic retinopathy,retinopathy of prematurity, and acute macular degeneration (wet type)).The compositions and methods of the present invention may be used totreat such conditions.

Experiments conducted during the course of development of embodiments ofthe present invention included fine mapping of anti-angiogoenic activityof the Pigment Epithelial-derived Factor, PEDF, using a 34-mer PEDFpeptide (amino acids 44-77 at the N-terminus). It was demonstrated thatan 18-mer fragment of PEDF effectively blocks VEGF-induced angiogenesisin vitro and in vivo in the subcutaneous matrigel plug assays.

This shorter PEDF fragment is more active in blocking angiogenesis thanthe original 34-mer. Accordingly, in some embodiments, the presentinvention provides PEDF peptides (e.g., the 18-mer; SEQ ID NO:2), orother short peptides (e.g., SEQ ID NOs: 3-49) for the treatment of theabove and other angiogenesis-dependent disease. In certain embodiments,the peptide is selected from an amino acid sequence represented by SEQID NOs: 2, 12, 13, 22, 23, 33, 34, 41-49, 52, 53, 62, 63, 73, 74, 84,85, and 92-100. In other embodiments, the peptide is selected from anamino acid sequence represented by SEQ ID NOs:1-49 and 52-100.

II. Therapeutic Applications

In some embodiments, the present invention provides therapies forangiogenic (e.g., cancer and ocular diseases) and neurovascular relateddiseases. In some embodiments, therapies provide PEDF peptides (e.g.,SEQ ID NO:1-49 or 52-100) for the treatment of angiogenic andneurovascular related diseases.

A. Administering Chemotherapeutics Comprising PEDF and/or PEDF Peptides

It is contemplated that PEDF, PEDF-derived peptides (e.g., SEQ IDNO:1-49 or 52-100), and PEDF-derived peptide analogues or mimetics, canbe administered systemically or locally to inhibit tumor cellproliferation and angiogenesis, and induce tumor cell death in cancerpatients. They can be administered intravenously, intrathecally,intraperitoneally as well as orally. Moreover, they can be administeredalone or in combination with anti-proliferative drugs.

i. PEDF Peptides

The present invention is not limited to a particular PEDF derivedpeptide. In some embodiments, the 18 amino acid peptide described by SEQID NO:2 or 52 is utilized. In other embodiments, shorter (e.g., 13, 14,15, 16, or 17) amino acid peptides may be utilized. For example,exemplary peptides include, but are not limited to, a peptide comprisingor consisting of one of the following sequences:

DLYRVRSSTSPTTN; (SEQ ID NO: 1) NFGYDLYRVRSSTSPTTN; (SEQ ID NO: 2)FGYDLYRVRSSTSPTTN; (SEQ ID NO: 4) GYDLYRVRSSTSPTTN; (SEQ ID NO: 5)YDLYRVRSSTSPTTN; (SEQ ID NO: 6) NFGYDLYRVRSSTSPTT; (SEQ ID NO: 7)NFGYDLYRVRSSTSPT; (SEQ ID NO: 8) NFGYDLYRVRSSTSP; (SEQ ID NO: 9)NFGYDLYRVRSSTS; (SEQ ID NO: 10) or NFGYDLYRVRSST. (SEQ ID NO: 11)

In other embodiments, peptides longer than the 18-mer shown in SEQ IDNO:2 (NFGYDLYRVRSSTSPTTN) are employed. For example, exemplary peptidesinclude, but are not limited to a peptide comprising or consisting ofone of the following sequences:

(SEQ ID NO: 12) SNFGYDLYRVRSSTSPTTN; (SEQ ID NO: 13)VSNFGYDLYRVRSSTSPTTN; (SEQ ID NO: 14) AVSNFGYDLYRVRSSTSPTTN;(SEQ ID NO: 15) AAVSNFGYDLYRVRSSTSPTTN; (SEQ ID NO: 3)AAAVSNFGYDLYRVRSSTSPTTN; (SEQ ID NO: 16) LAAAVSNFGYDLYRVRSSTSPTTN;(SEQ ID NO: 17) KLAAAVSNFGYDLYRVRSSTSPTTN; (SEQ ID NO: 18)NKLAAAVSNFGYDLYRVRSSTSPTTN; (SEQ ID NO: 19) VNKLAAAVSNFGYDLYRVRSSTSPTTN;(SEQ ID NO: 20) PVNKLAAAVSNFGYDLYRVRSSTSPTTN; (SEQ ID NO: 21)VPVNKLAAAVSNFGYDLYRVRSSTSPTTN; (SEQ ID NO: 22) NFGYDLYRVRSSTSPTTNV;(SEQ ID NO: 23) NFGYDLYRVRSSTSPTTNVL; (SEQ ID NO: 24)NFGYDLYRVRSSTSPTTNVLL; (SEQ ID NO: 25) NFGYDLYRVRSSTSPTTNVLLS;(SEQ ID NO: 26) NFGYDLYRVRSSTSPTTNVLLSP; (SEQ ID NO: 27)NFGYDLYRVRSSTSPTTNVLLSPL; (SEQ ID NO: 28) NFGYDLYRVRSSTSPTTNVLLSPLS;(SEQ ID NO: 29) NFGYDLYRVRSSTSPTTNVLLSPLSV; (SEQ ID NO: 30)NFGYDLYRVRSSTSPTTNVLLSPLSVA; (SEQ ID NO: 31)NFGYDLYRVRSSTSPTTNVLLSPLSVAT; (SEQ ID NO: 32)NFGYDLYRVRSSTSPTTNVLLSPLSVATA; (SEQ ID NO: 33) SNFGYDLYRVRSSTSPTTNV;(SEQ ID NO: 34) VSNFGYDLYRVRSSTSPTTNVL; (SEQ ID NO: 35)AVSNFGYDLYRVRSSTSPTTNVLL; (SEQ ID NO: 36) AAVSNFGYDLYRVRSSTSPTTNVLLS;(SEQ ID NO: 37) AAAVSNFGYDLYRVRSSTSPTTNVLLSP; (SEQ ID NO: 38)LAAAVSNFGYDLYRVRSSTSPTTNVLLSPL; (SEQ ID NO: 39)KLAAAVSNFGYDLYRVRSSTSPTTNVLLSPLS; (SEQ ID NO: 40)NKLAAAVSNFGYDLYRVRSSTSPTTNVLLSPLSV; (SEQ ID NO: 41)SNFGYDLYRVRSSTSPTTNVL; (SEQ ID NO: 42) VSNFGYDLYRVRSSTSPTTNV;(SEQ ID NO: 43) XNFGYDLYRVRSSTSPTTN; (SEQ ID NO: 44)XXNFGYDLYRVRSSTSPTTN; (SEQ ID NO: 45) NFGYDLYRVRSSTSPTTNX;(SEQ ID NO: 46) NFGYDLYRVRSSTSPTTNXX; (SEQ ID NO: 47)XNFGYDLYRVRSSTSPTTNX; (SEQ ID NO: 48) XNFGYDLYRVRSSTSPTTNXX; or(SEQ ID NO: 49) XXNFGYDLYRVRSSTSPTTNXX.

In the above sequences, “X” stands for any amino acid, includingmodified amino acids.

Peptides that are longer or short than SEQ ID NO:2 can be designed basedon a PEDF sequence (e.g., SEQ ID NO:50 or 101) shown in FIGS. 7 and 10respectively (e.g., by adding or deleting amino acids from either end ofSEQ ID NO:2 or 52).

In other embodiments, other PEDF peptides are employed (e.g., thatreplace a “T” with an “M” at position 13 in SEQ ID NO:2).

(SEQ ID NO: 52) DLYRVRSSMSPTTN; (SEQ ID NO: 53) NFGYDLYRVRSSMSPTTN;(SEQ ID NO: 54) FGYDLYRVRSSMSPTTN; (SEQ ID NO: 55) GYDLYRVRSSMSPTTN;(SEQ ID NO: 56) YDLYRVRSSMSPTTN; (SEQ ID NO: 57) NFGYDLYRVRSSMSPTT;(SEQ ID NO: 58) NFGYDLYRVRSSMSPT; (SEQ ID NO: 59) NFGYDLYRVRSSMSP;(SEQ ID NO: 60) NFGYDLYRVRSSMS; (SEQ ID NO: 61) NFGYDLYRVRSSM;(SEQ ID NO: 62) SNFGYDLYRVRSSMSPTTN; (SEQ ID NO: 63)VSNFGYDLYRVRSSMSPTTN; (SEQ ID NO: 64) AVSNFGYDLYRVRSSMSPTTN;(SEQ ID NO: 65) AAVSNFGYDLYRVRSSMSPTTN; (SEQ ID NO: 66)AAAVSNFGYDLYRVRSSMSPTTN; (SEQ ID NO: 67) LAAAVSNFGYDLYRVRSSMSPTTN;(SEQ ID NO: 68) KLAAAVSNFGYDLYRVRSSMSPTTN; (SEQ ID NO: 69)NKLAAAVSNFGYDLYRVRSSMSPTTN; (SEQ ID NO: 70) VNKLAAAVSNFGYDLYRVRSSMSPTTN;(SEQ ID NO: 71) PVNKLAAAVSNFGYDLYRVRSSMSPTTN; (SEQ ID NO: 72)VPVNKLAAAVSNFGYDLYRVRSSMSPTTN; (SEQ ID NO: 73) NFGYDLYRVRSSMSPTTNV;(SEQ ID NO: 74) NFGYDLYRVRSSMSPTTNVL; (SEQ ID NO: 75)NFGYDLYRVRSSMSPTTNVLL; (SEQ ID NO: 76) NFGYDLYRVRSSMSPTTNVLLS;(SEQ ID NO: 77) NFGYDLYRVRSSMSPTTNVLLSP; (SEQ ID NO: 78)NFGYDLYRVRSSMSPTTNVLLSPL; (SEQ ID NO: 79) NFGYDLYRVRSSMSPTTNVLLSPLS;(SEQ ID NO: 80) NFGYDLYRVRSSMSPTTNVLLSPLSV; (SEQ ID NO: 81)NFGYDLYRVRSSMSPTTNVLLSPLSVA; (SEQ ID NO: 82)NFGYDLYRVRSSMSPTTNVLLSPLSVAT; (SEQ ID NO: 83)NFGYDLYRVRSSMSPTTNVLLSPLSVATA; (SEQ ID NO: 84) SNFGYDLYRVRSSMSPTTNV;(SEQ ID NO: 85) VSNFGYDLYRVRSSMSPTTNVL; (SEQ ID NO: 86)AVSNFGYDLYRVRSSMSPTTNVLL; (SEQ ID NO: 87) AAVSNFGYDLYRVRSSMSPTTNVLLS;(SEQ ID NO: 88) AAAVSNFGYDLYRVRSSMSPTTNVLLSP; (SEQ ID NO: 89)LAAAVSNFGYDLYRVRSSMSPTTNVLLSPL; (SEQ ID NO: 90)KLAAAVSNFGYDLYRVRSSMSPTTNVLLSPLS; (SEQ ID NO: 91)NKLAAAVSNFGYDLYRVRSSMSPTTNVLLSPLSV; (SEQ ID NO: 92)SNFGYDLYRVRSSMSPTTNVL; (SEQ ID NO: 93) VSNFGYDLYRVRSSMSPTTNV;(SEQ ID NO: 94) XNFGYDLYRVRSSMSPTTN; (SEQ ID NO: 95)XXNFGYDLYRVRSSMSPTTN; (SEQ ID NO: 96) NFGYDLYRVRSSMSPTTNX;(SEQ ID NO: 97) NFGYDLYRVRSSMSPTTNXX; (SEQ ID NO: 98)XNFGYDLYRVRSSMSPTTNX; (SEQ ID NO: 99) XNFGYDLYRVRSSMSPTTNXX; or(SEQ ID NO: 100) XXNFGYDLYRVRSSMSPTTNXX.

In the above sequences, “X” stands for any amino acid, includingmodified amino acids.

The PEDF peptides may be obtained using any suitable method. Forexample, in some embodiments, peptides are produced recombinantly inhost cells. Thus, for example, a polypeptide encoding the desired PEDFpeptides may be included in any one of a variety of expression vectorsfor expressing a polypeptide. In some embodiments of the presentinvention, vectors include, but are not limited to, chromosomal,nonchromosomal and synthetic DNA sequences (e.g., derivatives of SV40,bacterial plasmids, phage DNA; baculovirus, yeast plasmids, vectorsderived from combinations of plasmids and phage DNA, and viral DNA suchas vaccinia, adenovirus, fowl pox virus, and pseudorabies). It iscontemplated that any vector may be used as long as it is replicable andviable in the host.

In some embodiments of the present invention, the constructs comprise avector, such as a plasmid or viral vector, into which a sequenceencoding a PEDF peptide has been inserted, in a forward or reverseorientation. In still other embodiments, the sequence is assembled inappropriate phase with translation initiation and termination sequences.In some embodiments, the appropriate DNA sequence is inserted into thevector using any of a variety of procedures. In general, the DNAsequence is inserted into an appropriate restriction endonucleasesite(s) by procedures known in the art.

Large numbers of suitable vectors are known to those of skill in theart, and are commercially available. Such vectors include, but are notlimited to, the following vectors: 1) Bacterial—pQE70, pQE60, pQE 9(Qiagen), pBS, pD10, phagescript, psiX174, pbluescript SK, pBSKS, pNH8A,pNH16a, pNH18A, pNH46A (Stratagene); ptrc99a, pKK223 3, pKK233 3,pDR540, pRIT5 (Pharmacia); and 2) Eukaryotic—pWLNEO, pSV2CAT, pOG44,PXT1, pSG (Stratagene) pSVK3, pBPV, pMSG, pSVL (Pharmacia). Any otherplasmid or vector may be used as long as they are replicable and viablein the host. In some preferred embodiments of the present invention,mammalian expression vectors comprise an origin of replication, asuitable promoter and enhancer, and also any necessary ribosome bindingsites, polyadenylation sites, splice donor and acceptor sites,transcriptional termination sequences, and 5′ flanking non transcribedsequences. In other embodiments, DNA sequences derived from the SV40splice, and polyadenylation sites may be used to provide the requirednon transcribed genetic elements.

In certain embodiments of the present invention, the DNA sequence in theexpression vector is operatively linked to an appropriate expressioncontrol sequence(s) (promoter) to direct mRNA synthesis. Promotersuseful in the present invention include, but are not limited to, the LTRor SV40 promoter, the E. coli lac or trp, the phage lambda PL and PR, T3and T7 promoters, and the cytomegalovirus (CMV) immediate early, herpessimplex virus (HSV) thymidine kinase, and mouse metallothionein Ipromoters and other promoters known to control expression of gene inprokaryotic or eukaryotic cells or their viruses. In other embodimentsof the present invention, recombinant expression vectors include originsof replication and selectable markers permitting transformation of thehost cell (e.g., dihydrofolate reductase or neomycin resistance foreukaryotic cell culture, or tetracycline or ampicillin resistance in E.coli).

In some embodiments of the present invention, transcription of the DNAencoding the polypeptides of the present invention by higher eukaryotesis increased by inserting an enhancer sequence into the vector.Enhancers are cis acting elements of DNA, usually about from 10 to 300by that act on a promoter to increase its transcription. Enhancersuseful in the present invention include, but are not limited to, theSV40 enhancer on the late side of the replication origin by 100 to 270,a cytomegalovirus early promoter enhancer, the polyoma enhancer on thelate side of the replication origin, and adenovirus enhancers.

In other embodiments, the expression vector also contains a ribosomebinding site for translation initiation and a transcription terminator.In still other embodiments of the present invention, the vector may alsoinclude appropriate sequences for amplifying expression.

In some embodiments, peptides are expressed in host cells. In someembodiments of the present invention, the host cell is a highereukaryotic cell (e.g., a mammalian or insect cell). In other embodimentsof the present invention, the host cell is a lower eukaryotic cell(e.g., a yeast cell). In still other embodiments of the presentinvention, the host cell can be a prokaryotic cell (e.g., a bacterialcell). Specific examples of host cells include, but are not limited to,Escherichia coli, Salmonella typhimurium, Bacillus subtilis, and variousspecies within the genera Pseudomonas, Streptomyces, and Staphylococcus,as well as Saccharomycees cerivisiae, Schizosaccharomycees pombe,Drosophila S2 cells, Spodoptera Sf9 cells, Chinese hamster ovary (CHO)cells, COS 7 lines of monkey kidney fibroblasts, (Gluzman, Cell 23:175[1981]), C127, 3T3, 293, 293T, HeLa and BHK cell lines.

The constructs in host cells can be used in a conventional manner toproduce the gene product encoded by the recombinant sequence. In someembodiments, introduction of the construct into the host cell can beaccomplished by transfection or electroporation (See e.g., Davis et al.,Basic Methods in Molecular Biology, [1986]). Alternatively, in someembodiments of the present invention, the polypeptides of the inventioncan be synthetically produced by conventional peptide synthesizers.

Peptides can be expressed in mammalian cells, yeast, bacteria, or othercells under the control of appropriate promoters. Cell free translationsystems can also be employed to produce such proteins using RNAs derivedfrom the DNA constructs of the present invention. Appropriate cloningand expression vectors for use with prokaryotic and eukaryotic hosts aredescribed by Sambrook, et al., Molecular Cloning: A Laboratory Manual,Second Edition, Cold Spring Harbor, N.Y., (1989).

In some embodiments of the present invention, following transformationof a suitable host strain and growth of the host strain to anappropriate cell density, the selected promoter is induced byappropriate means (e.g., temperature shift or chemical induction) andcells are cultured for an additional period. In other embodiments of thepresent invention, cells are typically harvested by centrifugation,disrupted by physical or chemical means, and the resulting crude extractretained for further purification. In still other embodiments of thepresent invention, microbial cells employed in expression of proteinscan be disrupted by any convenient method, including freeze thawcycling, sonication, mechanical disruption, or use of cell lysingagents.

Peptides may be recovered and purified from host cells, using anysuitable method include, but not limited to, ammonium sulfate or ethanolprecipitation, acid extraction, anion or cation exchange chromatography,phosphocellulose chromatography, hydrophobic interaction chromatography,affinity chromatography, hydroxylapatite chromatography and lectinchromatography. In some embodiments of the present invention, highperformance liquid chromatography (HPLC) is employed for purificationsteps.

In an alternate embodiment of the invention, nucleic acid sequenceencoding the peptides are synthesized, whole or in part, using chemicalmethods well known in the art (See e.g., Caruthers et al., Nucl. AcidsRes. Symp. Ser., 7:215 233 [1980]; Crea and Horn, Nucl. Acids Res.,9:2331 [1980]; Matteucci and Caruthers, Tetrahedron Lett., 21:719[1980]; and Chow and Kempe, Nucl. Acids Res., 9:2807 2817 [1981]). Inother embodiments of the present invention, the peptide is producedusing chemical methods. For example, peptides can be synthesized bysolid phase techniques, cleaved from the resin, and purified bypreparative high performance liquid chromatography (See e.g., Creighton,Proteins Structures And Molecular Principles, W H Freeman and Co, NewYork N.Y. [1983]). In some embodiments of the present invention, thecomposition of the synthetic peptides is confirmed by amino acidanalysis or sequencing (See e.g., Creighton, supra).

Direct peptide synthesis can be performed using various solid phasetechniques (Roberge et al., Science 269:202 204 [1995]) and automatedsynthesis may be achieved, for example, using ABI 431A PeptideSynthesizer (Perkin Elmer) in accordance with the instructions providedby the manufacturer.

ii. Preparations and Administration

Where combinations are contemplated, it is not intended that the presentinvention be limited by the particular nature of the combination. Thepresent invention contemplates combinations as simple mixtures as wellas chemical hybrids. An example of the latter is where the peptide ordrug is covalently linked to a targeting carrier or to an activepharmaceutical. Covalent binding can be accomplished by any one of manycommercially available crosslinking compounds.

It is not intended that the present invention be limited by theparticular nature of the therapeutic preparation. For example, suchcompositions can be provided together with physiologically tolerableliquid, gel or solid carriers, diluents, adjuvants and excipients.

These therapeutic preparations can be administered to mammals forveterinary use, such as with domestic animals, and clinical use inhumans in a manner similar to other therapeutic agents. In general, thedosage required for therapeutic efficacy will vary according to the typeof use and mode of administration, as well as the particularizedrequirements of individual hosts.

Such compositions are typically prepared as liquid solutions orsuspensions, or in solid forms. Oral formulations for cancer usuallywill include such normally employed additives such as binders, fillers,carriers, preservatives, stabilizing agents, emulsifiers, buffers andexcipients as, for example, pharmaceutical grades of mannitol, lactose,starch, magnesium stearate, sodium saccharin, cellulose, magnesiumcarbonate, and the like. These compositions take the form of solutions,suspensions, tablets, pills, capsules, sustained release formulations,or powders, and typically contain 1%-95% of active ingredient,preferably 2%-70%.

The compositions are also prepared as injectables, either as liquidsolutions or suspensions; solid forms suitable for solution in, orsuspension in, liquid prior to injection may also be prepared.

The compositions of the present invention are often mixed with diluentsor excipients which are physiological tolerable and compatible. Suitablediluents and excipients are, for example, water, saline, dextrose,glycerol, or the like, and combinations thereof. In addition, if desiredthe compositions may contain minor amounts of auxiliary substances suchas wetting or emulsifying agents, stabilizing or pH buffering agents.

Additional formulations which are suitable for other modes ofadministration, such as topical administration, include salves,tinctures, creams, lotions, and, in some cases, suppositories. Forsalves and creams, traditional binders, carriers and excipients mayinclude, for example, polyalkylene glycols or triglycerides.

B. Designing Mimetics

It may be desirable to administer an analogue of a PEDF-derived peptide(e.g., an analogue of SEQ ID NO:2 or SEQ ID NO:52). A variety of designsfor such mimetics are possible. For example, cyclic peptides, in whichthe necessary conformation for binding is stabilized by nonpeptides, arespecifically contemplated. (See, e.g., U.S. Pat. No. 5,192,746 to Loblet al., U.S. Pat. No. 5,169,862 to Burke, Jr. et al., U.S. Pat. No.5,539,085 to Bischoff et al., U.S. Pat. No. 5,576,423 to Aversa et al.,U.S. Pat. No. 5,051,448 to Shashoua, and U.S. Pat. No. 5,559,103 toGaeta et al., all hereby incorporated by reference, describe multiplemethods for creating such compounds.

Synthesis of nonpeptide compounds that mimic peptide sequences is alsoknown in the art. For example, Eldred et al., J. Med. Chem. 37:3882(1994), describe nonpeptide antagonists that mimic the Arg-Gly-Aspsequence. Likewise, Ku et al., J. Med. Chem. 38:9 (1995) give furtherelucidation of the synthesis of a series of such compounds. Suchnonpeptide compounds are specifically contemplated by the presentinvention.

The present invention also contemplates synthetic mimicking compoundsthat are multimeric compounds that repeat the relevant peptide sequence.As is known in the art, peptides can be synthesized by linking an aminogroup to a carboxyl group that has been activated by reaction with acoupling agent, such as dicyclohexyl-carbodiimide (DCC). The attack of afree amino group on the activated carboxyl leads to the formation of apeptide bond and the release of dicyclohexylurea. It may be important toprotect potentially reactive groups other than the amino and carboxylgroups intended to react. For example, the (x-amino group of thecomponent containing the activated carboxyl group can be blocked with atertbutyloxycarbonyl group. This protecting group can be subsequentlyremoved by exposing the peptide to dilute acid, which leaves peptidebonds intact.

With this method, peptides can be readily synthesized by a solid phasemethod by adding amino acids stepwise to a growing peptide chain that islinked to an insoluble matrix, such as polystyrene beads. Thecarboxyl-terminal amino acid (with an amino protecting group) of thedesired peptide sequence is first anchored to the polystyrene beads. Theprotecting group of the amino acid is then removed. The next amino acid(with the protecting group) is added with the coupling agent. This isfollowed by a washing cycle. The cycle is repeated as necessary.

The methods of the present invention can be practiced in vitro or invivo.

For example, the method of the present invention can be used in vitro toscreen for compounds which are potentially useful for combinatorial usewith PEDF peptides (e.g., SEQ ID NOs: 1-49 or 52-100) for treatingcancer or other angiogenic or neurovascular diseases; to evaluate acompound's efficacy in treating cancer; or to investigate the mechanismby which a compound combats cancer or other angiogenic or neurovasculardiseases (e.g., whether it does so by inducing apoptosis, by inducingdifferentiation, by decreasing proliferation, etc). For example, once acompound has been identified as a compound that works in combinationwith PEDF peptides to inhibit angiogenesis, proliferation and/or causeapoptosis of cancer cells, one skilled in the art can apply the methodof the present invention in vitro to evaluate the degree to which thecompound induces apoptosis and/or decreases angiogenesis, proliferationof cancer cells; or one skilled in the art can apply the method of thepresent invention to determine whether the compound operates by inducingapoptosis, by decreasing proliferation and/or angiogenesis, or by acombination of these methods.

Alternatively, the method of the present invention can be used in vivoto treat cancers, (e.g., including, but not limited to, ovarian cancer,breast cancer) or other angiogenic or neurovascular diseases. In thecase where the method of the present invention is carried out in vivo,for example, where the cancer cells are present in a human subject,contacting can be carried out by administering a therapeuticallyeffective amount of the compound to the human subject (e.g., by directlyinjecting the compound into a tumor or through systemic administration).

The present invention, in another aspect thereof, relates to a method oftreating cancer or other angiogenic or neurovascular diseases. Themethod includes administering to the subject an amount of a compoundeffective to inhibit angiogenesis, proliferation and/or cause the deathof cancer cells.

Suitable subjects include, for example mammals, such as rats, mice,cats, dogs, monkeys, and humans. Suitable human subjects include, forexample, those which have previously been determined to be at risk ofhaving cancer or other angiogenic or neurovascular diseases and thosewho have been diagnosed as having cancer or other angiogenic orneurovascular diseases.

In subjects who are determined to be at risk of having cancer, thecompositions of the present invention are administered to the subjectpreferably under conditions effective to decrease angiogenesis,proliferation and/or induce apoptosis of the cancer cells in the eventthat they develop.

The compositions herein may be made up in any suitable form appropriatefor the desired use. Examples of suitable dosage forms include oral,parenteral, or topical dosage forms.

Suitable dosage forms for oral use include tablets, dispersible powders,granules, capsules, suspensions, syrups, and elixirs. Inert diluents andcarriers for tablets include, for example, calcium carbonate, sodiumcarbonate, lactose, and talc. Tablets may also contain granulating anddisintegrating agents, such as starch and alginic acid; binding agents,such as starch, gelatin, and acacia; and lubricating agents, such asmagnesium stearate, stearic acid, and talc. Tablets may be uncoated ormay be coated by known techniques to delay disintegration andabsorption. Inert diluents and carriers which may be used in capsulesinclude, for example, calcium carbonate, calcium phosphate, and kaolin.Suspensions, syrups, and elixirs may contain conventional excipients,for example, methyl cellulose, tragacanth, sodium alginate; wettingagents, such as lecithin and polyoxyethylene stearate; andpreservatives, such as ethyl-p-hydroxybenzoate.

Dosage forms suitable for parenteral administration include solutions,suspensions, dispersions, emulsions, and the like. They may also bemanufactured in the form of sterile solid compositions which can bedissolved or suspended in sterile injectable medium immediately beforeuse. They may contain suspending or dispersing agents known in the art.Examples of parenteral administration are intraventricular,intracerebral, intramuscular, intravenous, intraperitoneal, rectal, andsubcutaneous administration.

In addition to PEDF peptides (e.g., SEQ ID NOs: 1-49 or 52-100), thesecompositions can include other active materials, particularly, activeswhich have been identified as useful in the treatment of cancers (e.g.,adenocarcinomas). These actives can be broad-based anti-cancer agents,such that they also are useful in treating more than one type of canceror they may be more specific (e.g., in a case where the other activematerial is useful for treating adenocarcinomas but not useful fortreating oral squamous cell carcinoma). The other actives can also havenon-anti-cancer pharmacological properties in addition to theiranti-cancer properties. For example, the other actives can haveanti-inflammatory properties, or, alternatively, they can have no suchanti-inflammatory properties.

It will be appreciated that the actual preferred amount of compositioncomprising PEDF peptide to be administered according to the presentinvention may vary according to the particular composition formulated,and the mode of administration. Many factors that may modify the actionof the compositions (e.g., body weight, sex, diet, time ofadministration, route of administration, rate of excretion, condition ofthe subject, drug combinations, and reaction sensitivities andseverities) can be taken into account by those skilled in the art.Administration can be carried out continuously or periodically withinthe maximum tolerated dose. Optimal administration rates for a given setof conditions can be ascertained by those skilled in the art usingconventional dosage administration tests.

C. Therapeutic Agents Combined or Co-Administered with PEDF Peptides

A wide range of therapeutic agents find use with the present invention.For example, any therapeutic agent that can be co-administered with PEDFpeptides (e.g., SEQ ID NOs:1-49 or 52-100), or associated with PEDF issuitable for use in the present invention.

In some embodiments, PEDF peptides described herein (e.g., SEQ ID NO:2or 52) are administered in combination with the ERT 44-mer fragment, ERTof PEDF.

Some embodiments of the present invention provide administering to asubject an effective amount of PEDF peptides (and enantiomers,derivatives, and pharmaceutically acceptable salts thereof) and at leastone anticancer agent (e.g., a conventional anticancer agent, such as,chemotherapeutic drugs, and/or radiation therapy).

Anticancer agent mechanisms suitable for use with the present inventioninclude, but are not limited to, agents that induce apoptosis, agentsthat induce/cause nucleic acid damage, agents that inhibit nucleic acidsynthesis, agents that affect microtubule formation, and agents thataffect protein synthesis or stability.

Classes of anticancer agents suitable for use in compositions andmethods of the present invention include, but are not limited to: 1)alkaloids, including, microtubule inhibitors (e.g., Vincristine,Vinblastine, and Vindesine, etc.), microtubule stabilizers (e.g.,Paclitaxel (Taxol), and Docetaxel, etc.), and chromatin functioninhibitors, including, topoisomerase inhibitors, such as,epipodophyllotoxins (e.g., Etoposide (VP-16), and Teniposide (VM-26),etc.), and agents that target topoisomerase I (e.g., Camptothecin andIsirinotecan (CPT-11), etc.); 2) covalent DNA-binding agents (alkylatingagents), including, nitrogen mustards (e.g., Mechlorethamine,Chlorambucil, Cyclophosphamide, Ifosphamide, and Busulfan (Myleran),etc.), nitrosoureas (e.g., Carmustine, Lomustine, and Semustine, etc.),and other alkylating agents (e.g., Dacarbazine, Hydroxymethylmelamine,Thiotepa, and Mitocycin, etc.); 3) noncovalent DNA-binding agents(antitumor antibiotics), including, nucleic acid inhibitors (e.g.,Dactinomycin (Actinomycin D), etc.), anthracyclines (e.g., Daunorubicin(Daunomycin, and Cerubidine), Doxorubicin (Adriamycin), and Idarubicin(Idamycin), etc.), anthracenediones (e.g., anthracycline analogues, suchas, (Mitoxantrone), etc.), bleomycins (Blenoxane), etc., and plicamycin(Mithramycin), etc.; 4) antimetabolites, including, antifolates (e.g.,Methotrexate, Folex, and Mexate, etc.), purine antimetabolites (e.g.,6-Mercaptopurine (6-MP, Purinethol), 6-Thioguanine (6-TG), Azathioprine,Acyclovir, Ganciclovir, Chlorodeoxyadenosine, 2-Chlorodeoxyadenosine(CdA), and 2′-Deoxycoformycin (Pentostatin), etc.), pyrimidineantagonists (e.g., fluoropyrimidines (e.g., 5-fluorouracil (Adrucil),5-fluorodeoxyuridine (FdUrd) (Floxuridine)) etc.), and cytosinearabinosides (e.g., Cytosar (ara-C) and Fludarabine, etc.); 5) enzymes,including, L-asparaginase, and hydroxyurea, etc.; 6) hormones,including, glucocorticoids, such as, antiestrogens (e.g., Tamoxifen,etc.), nonsteroidal antiandrogens (e.g., Flutamide, etc.), and aromataseinhibitors (e.g., anastrozole (Arimidex), etc.); 7) platinum compounds(e.g., Cisplatin and Carboplatin, etc.); 8) monoclonal antibodiesconjugated with anticancer drugs, toxins, and/or radionuclides, etc.; 9)biological response modifiers (e.g., interferons (e.g., IFN-γ, etc.) andinterleukins (e.g., IL-2, etc.), etc.); 10) adoptive immunotherapy; 11)hematopoietic growth factors; 12) agents that induce tumor celldifferentiation (e.g., all-trans-retinoic acid, etc.); 13) gene therapytechniques; 14) antisense therapy techniques; 15) tumor vaccines; 16)therapies directed against tumor metastases (e.g., Batimistat, etc.);and 17) other inhibitors of angiogenesis.

In preferred embodiments, the present invention provides administrationof an effective amount of PEDF peptides and at least one conventionalanticancer agent that induces apoptosis and/or prevents cancer cellproliferation to a subject. In some preferred embodiments, the subjecthas a disease characterized by metastasis. In yet other preferredembodiments, the present invention provides administration of aneffective amount of PEDF peptides and a taxane (e.g., Docetaxel) to asubject having a disease characterized by the overexpression of Bcl-2family protein(s) (e.g., Bcl-2 and/or Bcl-X_(L)).

The taxanes (e.g., Docetaxel) are an effective class of anticancerchemotherapeutic agents. (See e.g., K. D. Miller and G. W. Sledge, Jr.Cancer Investigation, 17:121-136 (1999)). While the present invention isnot intended to be limited to any particular mechanism, taxane-mediatedcell death is though to proceed through intercellular microtubulestabilization and subsequent induction of the apoptotic pathway. (Seee.g., S. Haldar et al., Cancer Research, 57:229-233 (1997)).

In some other embodiments, cisplatin and taxol are specificallycontemplated for use with the PEDF peptide compositions of the presentinvention. Cisplatin and Taxol have a well-defined action of inducingapoptosis in tumor cells (See e.g., Lanni et al., Proc. Natl. Acad.Sci., 94:9679 (1997); Tortora et al., Cancer Research 57:5107 (1997);and Zaffaroni et al., Brit. J. Cancer 77:1378 (1998)). However,treatment with these and other chemotherapeutic agents is difficult toaccomplish without incurring significant toxicity. The agents currentlyin use are generally poorly water soluble, quite toxic, and given atdoses that affect normal cells as wells as diseased cells. For example,paclitaxel (Taxol), one of the most promising anticancer compoundsdiscovered, is poorly soluble in water. Paclitaxel has shown excellentantitumor activity in a wide variety of tumor models such as the B 16melanoma, L1210 leukemias, MX-1 mammary tumors, and CS-1 colon tumorxenografts. However, the poor aqueous solubility of paclitaxel presentsa problem for human administration. Accordingly, currently usedpaclitaxel formulations require a cremaphor to solubilize the drug. Thehuman clinical dose range is 200-500 mg. This dose is dissolved in a 1:1solution of ethanol:cremaphor and diluted to one liter of fluid givenintravenously. The cremaphor currently used is polyethoxylated castoroil. It is given by infusion by dissolving in the cremaphor mixture anddiluting with large volumes of an aqueous vehicle. Direct administration(e.g., subcutaneous) results in local toxicity and low levels ofactivity.

Any pharmaceutical that is routinely used in a cancer therapy contextfinds use in the present invention. Conventional anticancer agents thatare suitable for administration with the disclosed PEDF peptidecompositions include, but are mot limited to, adriamycin,5-fluorouracil, etoposide, camptothecin, methotrexate, actinomycin-D,mitomycin C, or more preferably, cisplatin. These agent may be preparedand used as a combined therapeutic composition, or kit, by combining itwith an immunotherapeutic agent, as described herein.

In some embodiments of the present invention, the therapeutic PEDFtreatments further comprise one or more agents that directly cross-linknucleic acids (e.g., DNA) to facilitate DNA damage leading to asynergistic, antineoplastic agents of the present invention. Forexample, agents such as cisplatin, and other DNA alkylating agents maybe used. Cisplatin has been widely used to treat cancer, withefficacious doses used in clinical applications of 20 mg/M² for 5 daysevery three weeks for a total of three courses. The compositions of thepresent invention may be delivered via any suitable method, including,but not limited to, injection intravenously, subcutaneously,intratumorally, intraperitoneally, or topically (e.g., to mucosalsurfaces).

Agents that damage DNA also include compounds that interfere with DNAreplication, mitosis, and chromosomal segregation. Such chemotherapeuticcompounds include, but are not limited to, adriamycin, also known asdoxorubicin, etoposide, verapamil, podophyllotoxin, and the like. Thesecompounds are widely used in clinical settings for the treatment ofneoplasms, and are administered through bolus injections intravenouslyat doses ranging from 25-75 M/² at 21 day intervals for adriamycin, to35-50 Mg/M² for etoposide intravenously or double the intravenous doseorally.

Agents that disrupt the synthesis and fidelity of nucleic acidprecursors and subunits also lead to DNA damage and find use aschemotherapeutic agents in the present invention. A number of nucleicacid precursors have been developed. Particularly useful are agents thathave undergone extensive testing and are readily available. As such,agents such as 5-fluorouracil (5-FU) are preferentially used byneoplastic tissue, making this agent particularly useful for targetingto neoplastic cells. The doses delivered may range from 3 to 15mg/kg/day, although other doses may vary considerably according tovarious factors including stage of disease, amenability of the cells tothe therapy, amount of resistance to the agents and the like.

In preferred embodiments, the anticancer agents (e.g., anti-angiogenicfactors discussed herein) used in the present invention are those thatare amenable to co-administration with PEDF peptides or are otherwiseassociated with the PEDF peptides such that they can be delivered into asubject, tissue, or cell without loss of fidelity of anticancer effect.For a more detailed description of cancer therapeutic agents such as aplatinum complex, verapamil, podophyllotoxin, carboplatin, procarbazine,mechlorethamine, cyclophosphamide, camptothecin, ifosfamide, melphalan,chlorambucil, bisulfan, nitrosurea, adriamycin, dactinomycin,daunorubicin, doxorubicin, bleomycin, plicomycin, mitomycin, etoposide(VP16), tamoxifen, taxol, transplatinum, 5-fluorouracil, vincristin,vinblastin and methotrexate and other similar anti-cancer agents, thoseof skill in the art are referred to any number of instructive manualsincluding, but not limited to, the Physician's Desk reference and toGoodman and Gilman's “Pharmaceutical Basis of Therapeutics” ninthedition, Eds. Hardman et al., 1996.

In some embodiments, the drugs are attached to PEDF with photocleavablelinkers. For example, several heterobifunctional, photocleavable linkersthat find use with the present invention are described (See, e.g., Ottlet al., Bioconjugate Chem., 9:143 (1998)). These linkers can be eitherwater or organic soluble. They contain an activated ester that can reactwith amines or alcohols and an epoxide that can react with a thiolgroup. In between the two groups is a 3,4-dimethoxy6-nitrophenylphotoisomerization group, which, when exposed to near-ultraviolet light(365 nm), releases the amine or alcohol in intact form. Thus, thetherapeutic agent, when linked to the compositions of the presentinvention using such linkers, may be released in biologically active oractivatable form through exposure of the target area to near-ultravioletlight.

In an exemplary embodiment, an active group of a PEDF peptide is reactedwith the activated ester of the organic-soluble linker. This product inturn is reacted with the partially-thiolated surface of appropriatedendrimers (the primary amines of the dendrimers can be partiallyconverted to thiol-containing groups by reaction with asub-stoichiometric amount of 2-iminothiolano). Thus conjugated, the drugis inactive and will not harm normal cells. When the conjugate islocalized within tumor cells, it is exposed to laser light of theappropriate near-UV wavelength, causing the active drug to be releasedinto the cell.

An alternative to photocleavable linkers are enzyme cleavable linkers. Anumber of photocleavable linkers have been demonstrated as effectiveanti-tumor conjugates and can be prepared by attaching cancertherapeutics, such as doxorubicin, to water-soluble polymers withappropriate short peptide linkers (See e.g., Vasey et al., Clin. CancerRes., 5:83 (1999)). The linkers are stable outside of the cell, but arecleaved by thiolproteases once within the cell. In a preferredembodiment, the conjugate PK1 is used. As an alternative to thephotocleavable linker strategy, enzyme-degradable linkers, such asGly-Phe-Leu-Gly may be used.

The present invention is not limited by the nature of the therapeutictechnique. For example, other conjugates that find use with the presentinvention include, but are not limited to, using conjugated borondusters for BNCT (See, e.g., Capala et al., Bioconjugate Chem., 7:7(1996)), the use of radioisotopes, and conjugation of toxins such asricin.

Antimicrobial therapeutic agents may also be used in combination withPEDF peptides as therapeutic agents in the present invention. Any agentthat can kill, inhibit, or otherwise attenuate the function of microbialorganisms may be used, as well as any agent contemplated to have suchactivities. Antimicrobial agents include, but are not limited to,natural and synthetic antibiotics, antibodies, inhibitory proteins,antisense nucleic acids, membrane disruptive agents and the like, usedalone or in combination. Indeed, any type of antibiotic may be usedincluding, but not limited to, anti-bacterial agents, anti-viral agents,anti-fungal agents, and the like.

In still further embodiments, another component of the present inventionis that the PEDF peptide be associated with targeting agents (PEDFpeptide-targeting agent complex) that are able to specifically target aparticular cell type (e.g., tumor cell). Generally, the PEDF peptidethat is associated with a targeting agent, targets neoplastic cellsthrough interaction of the targeting agent with a cell surface moietyand is taken into the cell through receptor mediated endocytosis.

Any moiety known to be located on the surface of target cells (e.g.,tumor cells) finds use with the present invention. For example, anantibody directed against such a moiety targets the compositions of thepresent invention to cell surfaces containing the moiety. Alternatively,the targeting moiety may be a ligand directed to a receptor present onthe cell surface or vice versa. Similarly, vitamins also may be used totarget the therapeutics of the present invention to a particular cell.

In some embodiments of the present invention, the targeting moiety mayalso function as an agent to identify a particular tumor characterizedby expressing a receptor that the targeting agent (ligand) binds with,for example, tumor specific antigens including, but not limited to,carcinoembryonic antigen, prostate specific antigen, tyrosinase, ras, asialyly lewis antigen, erb, MAGE-1, MAGE-3, BAGE, MN, gp100, gp75, p97,proteinase 3, a mucin, CD81, CID9, CD63; CD53, CD38, CO-029, CA125, GD2,GM2 and O-acetyl GD3, M-TAA, M-fetal or M-urinary find use with thepresent invention. Alternatively the targeting moiety may be a tumorsuppressor, a cytokine, a chemokine, a tumor specific receptor ligand, areceptor, an inducer of apoptosis, or a differentiating agent.

Tumor suppressor proteins contemplated for targeting include, but arenot limited to, p16, p21, p27, p53, p73, Rb, Wilms tumor (WT-1), DCC,neurofibromatosis type 1 (NF-1), von Hippel-Lindau (VHL) disease tumorsuppressor, Maspin, Brush-1, BRCA-1, BRCA-2, the multiple tumorsuppressor (MTS), gp95/p97 antigen of human melanoma, renal cellcarcinoma-associated G250 antigen, KS 1/4 pan-carcinoma antigen, ovariancarcinoma antigen (CA125), prostate specific antigen, melanoma antigengp75, CD9, CD63, CD53, CD37, R2, CD81, CO029, TI-1, L6 and SAS. Ofcourse these are merely exemplary tumor suppressors and it is envisionedthat the present invention may be used in conjunction with any otheragent that is or becomes known to those of skill in the art as a tumorsuppressor.

In preferred embodiments of the present invention, targeting is directedto factors expressed by an oncogene (e.g., bcl-2 and/or bcl-X_(L)).These include, but are not limited to, tyrosine kinases, bothmembrane-associated and cytoplasmic forms, such as members of the Srcfamily, serine/threonine kinases, such as Mos, growth factor andreceptors, such as platelet derived growth factor (PDDG), SMALL GTPases(G proteins) including the ras family, cyclin-dependent protein kinases(cdk), members of the myc family members including c-myc, N-myc, andL-myc and bcl-2 and family members.

Receptors and their related ligands that find use in the context of thepresent invention include, but are not limited to, the folate receptor,adrenergic receptor, growth hormone receptor, luteinizing hormonereceptor, estrogen receptor, epidermal growth factor receptor,fibroblast growth factor receptor, and the like.

Hormones and their receptors that find use in the targeting aspect ofthe present invention include, but are not limited to, growth hormone,prolactin, placental lactogen, luteinizing hormone, foilicle-stimulatinghormone, chorionic gonadotropin, thyroid-stimulating hormone, leptin,adrenocorticotropin (ACTH), angiotensin I, angiotensin II,.alpha.-endorphin, .alpha. melanocyte stimulating hormone,cholecystokinin, endothelin I, galanin, gastric inhibitory peptide(GIP), glucagon, insulin, amylin, lipotropins, GLP-1 (7-37)neurophysins, and somatostatin.

In addition, the present invention contemplates that vitamins (both fatsoluble and non-fat soluble vitamins) used as targeting agents may beused to target cells that have receptors for, or otherwise take up thesevitamins. Particularly preferred for this aspect are the fat solublevitamins, such as vitamin D and its analogues, vitamin E, Vitamin A, andthe like or water soluble vitamins such as Vitamin C, and the like.

In some embodiments of the present invention, any number of cancer celltargeting groups are associated with PEDF peptides (e.g., SEQ IDNOs:1-49 or 52-100). Thus, PEDF peptides associated with targetinggroups are specific for targeting cancer cells (i.e., much more likelyto attach to cancer cells and not to healthy cells).

In preferred embodiments of the present invention, targeting groups areassociated (e.g., covalently or noncovalently bound) to PEDF peptideswith either short (e.g., direct coupling), medium (e.g., usingsmall-molecule bifunctional linkers such as SPDP, sold by PierceChemical Company), or long (e.g., PEG bifunctional linkers, sold byShearwater Polymers) linkages.

In preferred embodiments of the present invention, the targeting agentis an antibody or antigen binding fragment of an antibody (e.g., Fabunits). For example, a well-studied antigen found on the surface of manycancers (including breast HER2 tumors) is glycoprotein p185, which isexclusively expressed in malignant cells (Press et al., Oncogene 5:953(1990)). Recombinant humanized anti-HER2 monoclonal antibodies(rhuMabHER2) have even been shown to inhibit the growth of HER2overexpressing breast cancer cells, and are being evaluated (inconjunction with conventional chemotherapeutics) in phase III clinicaltrials for the treatment of advanced breast cancer (Pegrarn et al.,Proc. Am. Soc. Clin. Oncol., 14:106 (1995)). Park et al. have attachedFab fragments of rhuMabHER2 to small unilamellar liposomes, which thencan be loaded with the chemotherapeutic doxorubicin (dox) and targetedto HER2 overexpressing tumor xenografts (Park et al., Cancer Lett.,118:153 (1997) and Kirpotin et al., Biochem., 36:66 (1997)). Thesedox-loaded “immunoliposomes” showed increased cytotoxicity againsttumors compared to corresponding non-targeted dox-loaded liposomes orfree dox, and decreased systemic toxicity compared to free dox.

Antibodies can be generated to allow for the targeting of antigens orimmunogens (e.g., tumor, tissue or pathogen specific antigens) onvarious biological targets (e.g., pathogens, tumor cells, normaltissue). Such antibodies include, but are not limited to polyclonal,monoclonal, chimeric, single chain, Fab fragments, and an Fab expressionlibrary.

In some preferred embodiments, the antibodies recognize tumor specificepitopes (e.g., TAG-72 (Kjeldsen et al., Cancer Res. 48:2214-2220(1988); U.S. Pat. Nos. 5,892,020; 5,892,019; and 5,512,443); humancarcinoma antigen (U.S. Pat. Nos. 5,693,763; 5,545,530; and 5,808,005);TP1 and TP3 antigens from osteocarcinoma cells (U.S. Pat. No.5,855,866); Thomsen-Friedenreich (TF) antigen from adenocarcinoma cells(U.S. Pat. No. 5,110,911); “KC-4 antigen” from human prostrateadenocarcinoma (U.S. Pat. Nos. 4,708,930 and 4,743,543); a humancolorectal cancer antigen (U.S. Pat. No. 4,921,789); CA125 antigen fromcystadenocarcinoma (U.S. Pat. No. 4,921,790); DF3 antigen from humanbreast carcinoma (U.S. Pat. Nos. 4,963,484 and 5,053,489); a humanbreast tumor antigen (U.S. Pat. No. 4,939,240); p97 antigen of humanmelanoma (U.S. Pat. No. 4,918,164); carcinoma or orosomucoid-relatedantigen (CORA)(U.S. Pat. No. 4,914,021); a human pulmonary carcinomaantigen that reacts with human squamous cell lung carcinoma but not withhuman small cell lung carcinoma (U.S. Pat. No. 4,892,935); T and Tnhaptens in glycoproteins of human breast carcinoma (Springer et al.,Carbohydr. Res. 178:271-292 (1988)), MSA breast carcinoma glycoproteintermed (Tjandra et al., Br. J. Surg. 75:811-817 (1988)); MFGM breastcarcinoma antigen (Ishida et al., Tumor Biol. 10:12-22 (1989)); DU-PAN-2pancreatic carcinoma antigen (Lan et al., Cancer Res. 45:305-310(1985)); CA125 ovarian carcinoma antigen (Hanisch et al., Carbohydr.Res. 178:29-47 (1988)); YH206 lung carcinoma antigen (Hinoda et al.,Cancer J., 42:653-658 (1988)). Each of the foregoing references arespecifically incorporated herein by reference.

Various procedures known in the art are used for the production ofpolyclonal antibodies. For the production of antibody, various hostanimals can be immunized by injection with the peptide corresponding tothe desired epitope including but not limited to rabbits, mice, rats,sheep, goats, etc. In a preferred embodiment, the peptide is conjugatedto an immunogenic carrier (e.g., diphtheria toxoid, bovine serum albumin(BSA), or keyhole limpet hemocyanin (KLH)). Various adjuvants are usedto increase the immunological response, depending on the host species,including but not limited to Freund's (complete and incomplete), mineralgels such as aluminum hydroxide, surface active substances such aslysolecithin, pluronic polyols, polyanions, peptides, oil emulsions,keyhole limpet hemocyanins, dinitrophenol, and potentially useful humanadjuvants such as BCG (Bacille Calmette-Guerin) and Corynebacteriumparvum.

For preparation of monoclonal antibodies, any technique that providesfor the production of antibody molecules by continuous cell lines inculture may be used (See e.g., Harlow and Lane, Antibodies: A LaboratoryManual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.).These include, but are not limited to, the hybridoma techniqueoriginally developed by Kohler and Milstein (Kohler and Milstein, Nature256:495-497 (1975)), as well as the trioma technique, the human B-cellhybridoma technique (See e.g., Kozbor et al., Immunol. Today 4:72(1983)), and the EBV-hybridoma technique to produce human monoclonalantibodies (Cole et al., in Monoclonal Antibodies and Cancer Therapy,Alan R. Liss, Inc., pp. 77-96 (1985)).

In an additional embodiment of the invention, monoclonal antibodies canbe produced in germ-free animals utilizing recent technology (See e.g.,PCT/US90/02545). According to the invention, human antibodies may beused and can be obtained by using human hybridomas (Cote et al., Proc.Natl. Acad. Sci. U.S.A. 80:2026-2030 (1983)) or by transforming human Bcells with EBV virus in vitro (Cole et al., in Monoclonal Antibodies andCancer Therapy, Alan R. Liss, pp. 77-96 (1985)).

According to the invention, techniques described for the production ofsingle chain antibodies (U.S. Pat. No. 4,946,778; herein incorporated byreference) can be adapted to produce specific single chain antibodies.An additional embodiment of the invention utilizes the techniquesdescribed for the construction of Fab expression libraries (Huse et al.,Science 246:1275-1281 (1989)) to allow rapid and easy identification ofmonoclonal Fab fragments with the desired specificity.

Antibody fragments that contain the idiotype (antigen binding region) ofthe antibody molecule can be generated by known techniques. For example,such fragments include but are not limited to: the F(ab′)₂ fragment thatcan be produced by pepsin digestion of the antibody molecule; the Fab′fragments that can be generated by reducing the disulfide bridges of theF(ab′)₂ fragment, and the Fab fragments that can be generated bytreating the antibody molecule with papain and a reducing agent.

In the production of antibodies, screening for the desired antibody canbe accomplished by techniques known in the art (e.g., radioimmunoassay,ELISA (enzyme-linked immunosorbant assay), “sandwich” immunoassays,immunoradiometric assays, gel diffusion precipitin reactions,immunodiffusion assays, in situ immunoassays (using colloidal gold,enzyme or radioisotope labels, for example), Western Blots,precipitation reactions, agglutination assays (e.g., gel agglutinationassays, hemagglutination assays, etc.), complement fixation assays,immunofluorescence assays, protein A assays, and immunoelectrophoresisassays, etc.).

For breast cancer, the cell surface may be targeted with folic acid,EGF, FGF, and antibodies (or antibody fragments) to the tumor-associatedantigens MUC1, cMet receptor and CD56 (NCAM).

A very flexible method to identify and select appropriate peptidetargeting groups is the phage display technique (See e.g., Cortese etal., Curr. Opin. Biotechol., 6:73 (1995)), which can be convenientlycarried out using commercially available kits. The phage displayprocedure produces a large and diverse combinatorial library of peptidesattached to the surface of phage, which are screened against immobilizedsurface receptors for tight binding. After the tight-binding, viralconstructs are isolated and sequenced to identify the peptide sequences.The cycle is repeated using the best peptides as starting points for thenext peptide library. Eventually, suitably high-affinity peptides areidentified and then screened for biocompatibility and targetspecificity. In this way, it is possible to produce peptides that can beconjugated to dendrimers, producing multivalent conjugates with highspecificity and affinity for the target cell receptors (e.g., tumor cellreceptors) or other desired targets.

Related to the targeting approaches described above is the“pretargeting” approach (See e.g., Goodwin and Meares, Cancer (suppl.)80:2675 (1997)). An example of this strategy involves initial treatmentof the patient with conjugates of tumor-specific monoclonal antibodiesand streptavidin. Remaining soluble conjugate is removed from thebloodstream with an appropriate biotinylated clearing agent. When thetumor-localized conjugate is all that remains, a gossypol-linked,biotinylated agent is introduced, which in turn localizes at the tumorsites by the strong and specific biotin-streptavidin interaction.

In some embodiments of the present invention, the targeting agents(moities) are preferably nucleic acids (e.g., RNA or DNA). In someembodiments, the nucleic acid targeting moities are designed tohybridize by base pairing to a particular nucleic acid (e.g.,chromosomal DNA, mRNA, or ribosomal RNA). In other embodiments, thenucleic acids bind a ligand or biological target. Nucleic acids thatbind the following proteins have been identified: reverse transcriptase,Rev and Tat proteins of HIV (Tuerk et al., Gene, 137(1):33-9 (1993));human nerve growth factor (Binkley et al., Nuc. Acids Res.,23(16):3198-205 (1995)); and vascular endothelial growth factor(Jellinek et al., Biochem., 83(34):10450-6 (1994)). Nucleic acids thatbind ligands are preferably identified by the SELEX procedure (See e.g.,U.S. Pat. Nos. 5,475,096; 5,270,163; and 5,475,096; and in PCTpublications WO 97/38134, WO 98/33941, and WO 99/07724, all of which areherein incorporated by reference), although many methods are known inthe art.

EXPERIMENTAL

The following examples are provided in order to demonstrate and furtherillustrate certain preferred embodiments and aspects of the presentinvention and are not to be construed as limiting the scope thereof.

EXAMPLE 1

This example describes the mapping of a 34-mer peptide of PEDF and theidentification of a shorter fragment, the 18-mer, with superioranti-angiogenic characteristics. FIG. 1 shows a schematic representationof identified functional domains of PEDF, including majorphosphorylation sites and domains known to be critical for interactionswith extracellular matrix. The analysis of the 34-mer structure wasperformed using Protean software and its hydrophilic profile, antigenicindices and probability of surface exposure were determined. Thisanalysis yielded several candidate peptides to be tested in the in vitroand in vivo angiogenesis assays (FIG. 2).

The C-terminal part of 34-mer is more hydrophilic, with a highly chargedarea in the middle and high antigenic index and thus is likely to beinvolved in the interaction with the putative receptor. Three peptideswere identified that cover this area as shown in FIG. 2: a shorter14-amino acid peptide that covers positively charged area with highlikelihood of surface exposure (DLYRVRSSTSPTTN; SEQ ID NO:1) and twoextended versions, one containing the negatively charged area(NFGY)(NFGYDLYRVRSSTSPTTN; SEQ ID NO:2), and the more extended one, thatcovers the adjacent neutral stretch (AAAV) (AAAVSNFGYDLYRVRSSTSPTTN; SEQID NO:3), which may ensure proper folding. The N-terminal sub-fragmentwas unlikely to be exposed on the surface of the molecule, and thereforewas not tested. Peptide synthesis has been performed to order atBioSource Custom Antibodies and Peptides.

The initial testing was done in the endothelial cell chemotaxis assay.Human microvascular endothelial cells to traverse gelatinizedmicroporous membrane up the gradient of bFGF was tested in the absenceand in the presence of the 14-, 18- and 23-mer peptides (FIG. 3). Humanmicrovascular endothelial cells were plated on the lower side ofgelatinized microporous membrane and allowed to adhere. The peptides(14-, 18 and 23-mer) were added at increasing concentrations to theopposite side of the gelatinized microporous membrane (8 μM pores),alone, or in combination with basic fibroblast growth factor (bFGF, 10ng/ml). The cells migratied to the opposite side of the membrane werecounted in 10 high-powered (100×) fields. Every condition was tested inquadruplicate. bFGF-induced chemotaxis is shown (top line in eachgraph). Basal migration levels (0.1% bovine serum albumin) are indicatedwith the dotted line. Triangles (Δ) show peptide alone; while squares(▪) show peptide with bFGF. The solid line (below the top line mentionedabove) indicates the effect of the recombinant human PEDF (rhPEDF) at 20nM.

The experiments showed that the 14-mer and 18-mer inhibited bFGF-inducedmigration in a dose-dependent manner. The 23-mer was comparable to PEDFat low concentrations and lost its inhibitory activity at higher doses.The inhibitor activity of the 14-mer and 18-mer were superior to that ofnative PEDF.

The ability of the 34-mer fragments to induce endothelial cell apoptosiswas next measured (FIG. 4). The 23-mer failed to induced apoptosis andthe 14-mer showed very weak induction of apoptosis. The 18-mer, however,was more potent than the parental PEDF and as potent as the 34-mer ininducing endothelial cell apoptosis.

Finally, the peptides were tested in vivo, in the corneal angiogenesisand directed in vivo angiogenesis assay (DIVAA). The 34-mer derivativeswere compared in vivo with parental PEDF peptide, the 34-mer. FIG. 5 A,B: Corneal angiogenesis assay: the peptides were incorporated with bFGF(50 ng/pellet) into slow-release sucralfate pellets, which weresurgically mplanted into the cornea of anesthetized mice, 0.5-1 mm fromthe vascular limbus. The responses were scored on day 5 postimplantation and the ingrowth of blood vessels from the cornea to thepellet was considered a positive response. The responses were scored aspositive corneas of total implanted: statistical significance wasevaluated with Fisher's Exact test. P<0.05 was considered significant.5A shows photographs of representative corneas. 5B shows the tabulatedresults of the cornea assay. 5C, D. DIVAA of the 34-mer and derivativepeptides. The peptides were incorporated with a mix of bFGF and VEGF(37.5 and 12.5 ng/ml, respectively) into angioreactors filled withmatrigel. The reactors were implanted s.c onto the flanks of the nudemice. On day 7, the reactors were harvested and photographed (5C).Endothelial cells collected from implants by dilution/centrifugation,stained with FITC-lectin and quantified by flow cytometry (5D). In bothassays the 18-mer showed the best anti-angiogenic characteristics (FIG.5). The 23-mer failed to inhibit angiogenesis in the DIVAA assay.

When tested in a tumorigenicity assay, the 18-mer significantlyinhibited the growth of PC-3 prostate cancer xenografts at 10 mg/kg:synthetic 34-mer had only marginal effect at this dose (FIG. 6). PC-3cells (2×106/site) were implanted into hindquarters of nude mice. On day5 post injection, treatment was commenced with 34-mer and 18-mer (10mg/kg, i.p.). Note substantial reduction in tumor volume in 18-mertreated tumors (FIG. 6).

EXAMPLE 2 P18 Suppresses the Growth of Renal Cell Carcinoma andMetastases in Orthtopic Xenigraft Model and Cooperates with Rapamycin

Mouse renal carcinoma cells (Renca, ATCC) were injected under the kidneycapsule (0.5×106/site). Animals were randomly divided into 6 groups (8per group) and the treatment started 5 days after inoculation(intraperitoneal injections). The following treatment groups wereincluded: P18 (SEQ ID NO:2; 10 mg/kg); P18 (60 mg/kg); Rapamycin (R, 0.5mg/kg); R+P18 (10 mg/kg) and R+P18 (60 mg/kg). The control group wastreated with scrambled peptide, YFNGRSSPSNTNTYYVDRL (SEQ ID NO:51).After 17 days of treatment both kidneys were removed. To assess tumorgrowth, the weight of healthy kidney was subtracted from the weight oftumor-inoculated kidney, for each animal. The difference represents theweight of the tumor. Average difference with SD meanings per group isshown in FIG. 8. Note significant (P<0.0003) decrease in tumor weight inP18-treated animals. Also note that the combination of Rapa (0.5 mg/kg)and P18 (60 mg/kg) completely abolished tumor growth (no difference fromthe basal variations in kidney weight (P=0.12). In the figure, thecontrol peptide is the scrambled peptide, (SEQ ID NO:51), P18 is the18-mer peptide (SEQ ID NO:2), and R is rapamycin.

Metastases formation was also examined in the liver and lungs of the inthe control treated animals and the groups treated with P18 andscrambled peptide (FIG. 9). The % affected organs is indicated under therepresentative images. Note that P18 treatment eradicated livermetastases, while combination treatment abolished lung metastases.

All publications and patents mentioned in the above specification areherein incorporated by reference as if expressly set forth herein.Various modifications and variations of the described methods andcompositions of the invention will be apparent to those skilled in theart without departing from the scope and spirit of the invention.Although the invention has been described in connection with specificpreferred embodiments, it should be understood that the invention asclaimed should not be unduly limited to such specific embodiments.Indeed, various modifications of the described modes for carrying outthe invention that are obvious to those skilled in relevant fields areintended to be within the scope of the invention.

1. A method of inhibiting angiogenesis, comprising: contacting a tissueexhibiting angiogenesis with a composition comprising an isolatedpeptide under conditions such that angiogenesis is decreased in saidtissue, wherein said peptide consists of an amino acid sequence selectedfrom the group consisting of: SEQ ID NOs:2, 12, 13, 22, 23, 33, 34, 41,42, 52, 53, 62, 63, 73, 74, 84, and
 85. 2. The method of claim 1,wherein said peptide consists of the amino acid sequence shown in SEQ IDNO:2 or SEQ ID NO:52.
 3. The method of claim 2, wherein said compositionfurther comprises a fluorescent compound.
 4. The method of claim 1,wherein said composition further comprises an anti-cancer agentdifferent from said peptide.
 5. The method of claim 4, wherein saidanti-cancer agent comprises rapamycin.
 6. The method of claim 1, whereinsaid composition further comprises a physiological tolerable buffer. 7.The method of claim 1, wherein said composition further comprises ananti-angiogenic agent different from said peptide.
 8. The method ofclaim 1, further comprising the step of administering a second agent tosaid tissue.
 9. The method of claim 1, wherein said tissue is canceroustissue.
 10. The method of claim 1, wherein said tissue is in a subject.